TRAF6 and NFAT seem to have similar functional roles in a variety of cell types, including T cells; leading to the possibility that in addition to regulation of NF-kappaB and MAP kinases, TRAF6 may function as well in the regulation of NFAT. I have shown that loss of TRAF6 abrogates NFAT activation in MEFs and that NFAT and TRAF6 associate in cells. This proposal is designed to expand the hypothesis that TRAF6 regulates NFAT, to examine the mechanism of this regulation, and to determine its physiological consequences in T cells. In Specific Aim 1, I will examine NFAT regulation and NFAT-dependent gene expression in TRAF6-/- mice. In Specific Aim 2, I will examine the functional effects of the interaction between TRAF6 and NFAT by creating point mutants of NFAT and TRAF6 that disrupt the interaction. The loss-of-association mutants will be used in knockout cells to specifically examine the role of the interaction in NFAT regulation and in functional activation of T cells. In Specific Aim 3, I will determine the role of TRAF6 ubiquitin E3 ligase activity in NFAT regulation. TRAF6 E3 ligase activity will be blocked by inhibiting the E2 ligase, Ubc13, in T cells and NFAT activation will then be examined.